Upregulation of apical NHE3 in renal OK cells overexpressing the rodent α1-subunit of the Na+pump

Abstract

Vectorial Na+reabsorption across the proximal tubule is mediated by apical entry of Na+, primarily via Na+/H+exchanger isoform 3 (NHE3), and basolateral extrusion via the Na+pump (Na+-K+-ATPase). We hypothesized that regulation of Na+reabsorption should involve not only the activity of the basolateral Na+-K+-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na+-K+-ATPase, opossum kidney (OK) cells were transfected with the rodent Na+-K+-ATPase α1-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na+-K+-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na+-K+-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC50values for ouabain: the first value was similar to the IC50of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum α1-isozymes. It is shown that transfection of OK cells with Na+-K+-ATPase increased Na+-K+-ATPase and NHE3 activities. This was associated with overexpression of the Na+-K+-ATPase α1-subunit and NHE3 in transfected OK cells. The abundance of the Na+-K+-ATPase β1-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na+-K+-ATPase in cells transfected with the rodent Na+pump α1-subunit cDNA is expected to stimulate apical Na+influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.